International Journal of Clinical Trials Aslam MN et al. Int J Clin Trials. 2020 Aug;7(3):170-175 http://www.ijclinicaltrials.com pISSN 2349-3240 | eISSN 2349-3259

DOI: http://dx.doi.org/10.18203/2349-3259.ijct20203103 Original Research Article Efficacy and safety of Prunus mume and choline in patients with abnormal level of liver function test

Muhammad N. Aslam1, Anwar Ali2, Suresh Kumar3*

1Department of Gastroenterology, Mayo Hospital, Lahore, Pakistan 2Department of Medicine, Saidu Group of Teaching Hospital, Khyber Pakhtunkhwa, Pakistan 3Department of Medicine, Rafa-e-Aam Hospital, Sindh, Pakistan

Received: 18 March 2020 Revised: 27 April 2020 Accepted: 30 April 2020

*Correspondence: Dr. Suresh Kumar, E-mail: [email protected]

Copyright: © the author(s), publisher and licensee Medip Academy. This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Background: The objectives of the study were to determine the efficacy and safety of Prunus mume and choline in patients with abnormal liver function test. Methods: This open labelled, multi-centered observational study was done for a from May 2019 to December 2019. Patients of either gender, above 18 years of age having elevated levels of aspartate aminotransferase, alanine transaminase or gamma-glutamyltransferase were included in the study after taking informed consent. One to two tablets of revolic per day were given preferably in the morning with breakfast or as per instructions of the physician. Patient follow-ups were done at 2nd and 4th week after treatment. SPSS version 20.0 was used for analysis. Frequency and percentages and for quantitative data, mean, standard deviation, median and interquartile range were recorded. Wilcoxon signed ranks test was applied with p value of <0.05 as significant level. Results: Among 247 patients, male to female ratio was 2:1 with overall mean age of 42.8±12.6 years. Total bilirubin decreased from baseline to week-4 in treatment with Prunus mume extract and choline (p=0.04). Median and IQR of alanine transaminase levels also reduced substantially from 99 (52) to 42 (36.5) I/U. Aspartate aminotransferase levels significantly decreased from 78.5 (57) to 40 (29) I/U. Overall at the end of treatment on week-4, 26 (10.5%) patients experienced gastrointestinal distress, 25 (10.1%) anorexia, 14 (5.7%) excessive salivation and 29 (11.7%) patients experienced excessive sweating.

Conclusions: This study reported significant improvement in alanine transaminase and aspartate aminotransferase levels after treatment with Prunus mume extract and choline (revolic). It is safe and effective to use them for deranged

liver functions tests.

Keywords: Prunus mume extract, Choline, Liver function tests

INTRODUCTION cause of chronic liver disease.2 Other lifestyle-associated risk factors include diabetes, obesity, smoking and non- Among various hepatic disorders, the most commonly alcoholic steato-hepatitis. Although substantial observed liver diseases are non-alcoholic fatty liver improvements have been made in treatment modalities disease, steato-hepatitis and hepatocellular carcinoma.1 through medications, yet the mortality rates have not Even though multiple causes of liver diseases have been shown significant improvements and so therefore, newer associated with it, infection with hepatitis B and hepatitis therapeutic strategies are the utmost need for putting an C especially in under-developed and developing countries end to such devastating disease.3 is highest. In developed world, alcohol is the leading

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Until recently, oxidative stress was reported to be one of phospholipids or are oxidized and used as methyl group the main risk factors for liver disease.4 Many herbal donor.14 The hepatic metabolite of choline is extracts having anti-oxidant activity like allium sativum, phosphatidylcholine, which is essentially important for glycyrrhizaglabra, taxacumofficinale, silybummarianum packaging, export of triglycerides in very low density and cichoriumintybus, all are studies for the beneficial lipoprotein and for bile salt solubility to be secreted.15 effect on a variety of diseases including hepatic disorders.5 Prunus mume, originating from south-eastern Multiple studies have been conducted outside the country China, is known as an anti-inflammatory and anti- but there is little data in Pakistan to support for the oxidative fruit. Extract of Prunus mume is an herbal effectiveness of Prunus mume and choline. Therefore, the variant that has been reported to exert a potentially objective of this study was to determine the safety and therapeutic effect for various conditions such as efficacy of Prunus mume and choline in patients with inflammatory bowel disease, diabetes mellitus, vascular abnormal level of liver function tests. dementia and cancers, including hepato-cellular carcinoma.6 METHODS

Prunus mume is regarded to contain 5-O-caffeoylquinic This open labeled, multi-centered observational study acidand 3-O-caffeoylquinic acid, both found to be a was done for a period of 06 months from May 2019 to major bioactive anti-oxidant compound.7 Prunus mume December 2019 by using convenient sampling technique. has been observed to be an effective treatment modality The study was conducted in Mayo Hospital, Lahore, in alcoholic liver disease having elevations in serum as Saidu Group of Teaching Hospital, Swat and Rafa-e-Aam well as hepatic triglycerides where serum triglycerides Hospital, Karachi. Using openEpi software, the sample were reported to reduce in treatment with Prunus mume. size calculator version 3.01 was used and using 47% It could be used for preventing alcoholic liver steatosis. efficacy of drug, at 07% margin of error and 95% Additionally, Prunus mume was seen to reduce cellular confidence interval, a sample size of 196 was calculated. oxidative stress through inhibition of mitogen-activated Total of 247 patients were included in the study.16 protein kinase’s activation and p-53 mediated apoptotic axis of signaling. This resulted in inhibiting hepatic After taking written informed consent and the study will steatosis and apoptosis as well.8 be carried out in compliance with the protocols and the principles of the Declaration of Helsinki, and in The constituents of Prunus mume, also known as accordance with the International Conference on Japanese apricot included triterpenoids, like oleanolic Harmonization harmonized tripartite guideline for good acid and ursolic acid, which has reported to inhibit clinical practice. Patients of either gender, above 18 years cellular growth and induce multiple tumors’ cell death, of age having elevated levels of aspartate such as in gastric cancer, pro-myelocytic leukemia, aminotransferase (AST), alanine transaminase (ALT) or pancreatic cancer, breast cancer, colon cancer, malignant gamma-glutamyl transferase (GGT), were included in the myeloma, esophageal cancer, lung cancer and hepato- study. Patients with obstructive jaundice, pregnant and cellular cancer.9 Lately, the hepato-protective effects of lactating mothers, having presence of underlying chronic Prunus mume have been observed in patients among renal failure were excluded from the study. chronic liver disease patients.10 Patients were given one to two tablets revolic per day, Since choline is used in synthesis of acetylcholine, a preferably in the morning with breakfast or as per neurotransmitter as well as involved in methyl-group instructions of the physician. The changes of serum ALT metabolism, especially in the liver. Therefore, the major and AST at baseline, 2nd week and 04th week of treatment site of choline metabolism is the liver, were it is mainly were noted after treatment with Prunus mume and found as phosphatidylcholine.11 It is associated with choline. Similarly, changes in (GGT) were also noted. assembly / secretion of lipoproteins and to solubilize The proportion of patients experiencing any adverse cholesterol in bile. The link between choline deficiency event such as gastrointestinal distress, anorexia, and accumulation of hepatic lipid has been recognized for salivation and sweating after taking the tablets were noted over 50 years, leading to the fact that choline-deficient and reported. These study parameters and sample size diets ought to induce non-alcoholic fatty liver disease. was selected in accordance with the study performed by Little is known regarding choline’s role in potentially Beretta et al in 2016.16 preventing or in treating non-alcoholic fatty liver disease.12,13 SPSS version 20.0 was used for analysis of data. For qualitative data, frequency in percentages was reported Choline is a content of cell membrane, mitochondrial and for quantitative data, mean, standard deviation, membrane and also an important component of median and interquartile range was recorded. Wilcoxon neurotransmitter, acetylcholine. Due to its essential signed ranks test was applied to test for significant ubiquitous presence in cellular components and difference at baselineweek-02 and week-04 keeping p- pathways, it is regarded as a main nutrient in metabolism value of <0.05 as statistically significant. and through phosphorylation are used to producing

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RESULTS The median and IQR (inter-quartile range) of total bilirubin was 1.1(2.3) mg/dl at week-2 (p=0.04). ALT In a total of 247 patients, 170 (69.1%) male and 77 was 99 (52) I/U at baseline, 61 (43) mg/dl at week-2 (30.9%) were included in the study. Patients were divided (p<0.01) and 42 (36.5) mg/dl at week-4 (p<0.01). AST into age groups with most common age group of above was 78.50 (57) I/U at baseline, 50 (32) mg/dl at week-2 50 years with the frequency of 68 (27.5%). Mean age of (p<0.01) and 40(36.5) mg/dl at week-4 (p<0.01). GGT patients was 42.8±12.6 years and weight were 78.1±18.4 was 40 (100.5) I/U at baseline, 18 (06) mg/dl at week-2 kg. 16 (8%) patients were diagnosed as hepatitis A, 08 (p=0.31) and 30 (97.5) mg/dl at week-4 (p=0.12) (Table (4%) as hepatitis B, 23 (11.6%) as hepatitis C, 11 (5.6%) 2, Figure 1). as non-specific hepatitis, 5 (2.5%) as auto-immune hepatitis and 135 (68%) as non-alcoholic fatty liver Overall, the end of treatment on week-4, 26 (10.5%) disease. 44 (18%) patients were diabetic, 55 (22%) patients experienced gastrointestinal distress, 25 (10.1%) hypertensive, 64 (26%) having hyperlipidemia, 57 (23%) anorexia, 14 (5.7%) excessive salivation and 29 (11.7%) and 78 (32%) obesity (Table 1). patients experienced excessive sweating.

Table 1: Baseline characteristics of studied samples (n=247).

Characteristics N % Male 170 69.1 Gender Female 77 30.9 16-30 56 22.6 31-40 57 23.0 Age group (in years) 41-50 66 26.7 >50 68 27.5 Mean±SD 42.8±12.6 Weight (kg) Mean±SD 78.1±18.4 Hepatitis A 16 8.1 Hepatitis B 8 4.0 Hepatitis C 23 11.6 Diagnosis Nonspecific hepatitis 11 5.6 Autoimmune hepatitis 5 2.5 NAFLD 135 68.2 Diabetes 44 17.8 High blood pressure 55 22.3 Co-morbidities High cholesterol 64 25.9 Smoking 57 23.1 Obesity 78 31.6

250

40 200

78.5

150 Gama GT 18 AST

Median 50 30 100 99 ALT 40 61 50 42

0 Day-o Week-2 Week-4

Figure 1: The level of ALT, AST and gamma GT at baseline, 2nd and 4th week.

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Table 2: Median comparison of parameters from baseline to week 4.

Baseline Week 2 Week 4 P value Parameters Baselines Baselines Median IQR Median IQR Median IQR week 2 week4 Total bilirubin 0.75 0.8 1.1 2.3 0.79 0.4 0.04* 0.11 Direct bilirubin 0.24 0.1 0.7 0.9 0.24 0.1 0.18 1.0 Indirect bilirubin 0.40 0.28 0.3 0.3 0.50 0.3 0.31 0.70 ALT 99.0 52 61.0 43.0 42.0 36.5 <0.01* <0.01* AST 78.50 57 50.0 32.0 40.0 29.0 <0.01* <0.01* Gama GT 40.0 100.5 18.0 6.0 30.0 97.5 0.31 0.12 *p<0.05 was considered significant using Wilcoxon signed ranks test.

DISCUSSION (p<0.01) were reported at both baseline and week 4. With regards to the observed side effects, majority of the In addition to hepato-protective effects, Prunus mume has patients were free of side effects while the patients who been reported to have anti-inflammatory and anti-oxidant experienced side effects, they were more or less tolerable. affects both in-vitro as well as in-vivo.17 In studies where 11.7% experienced sweating, 10.5% GI upset, 10% induced hepatotoxicity having high ALT and AST levels, anorexia while 5.7% salivation. Prunus mume was reported to substantially reduce lipid peroxidation and levels of malondialdehyde within liver In line with our study, the hepato-protective effect of PM tissues, thereby reducing the enzymatic levels of ALT among 58 patients with liver disease of hepatitis C, non- and AST.18 Prunus mume is reported to exert hepato- alcoholic fatty liver disease and auto-immune liver protective effect in both acute, induced liver injury as disease reported that after 12 weeks of treatment, serum well as in chronic hepatic fibrosis and cirrhosis. The ALT and AST levels dramatically decreased in proposed pathway by which Prunus mume works is comparison with baseline levels.18 attributed to enzymatic suppression, leading to reduction in hepatic damage as well as increasing anti-oxidant Similarly, Nagi et al Inappropriate, reported can be used chemicals which exert an even more hepato-protective and only reported in their study on 49 mice divided into effect on liver mitochondria.19 07 groups reported AST of 78±2.64 I/U and ALT of 126±1.00 I/U at baseline was reduced to 54±2.64 I/U and Similarly, choline is regarded as an essential nutrient 86±3.60 I/U respectively, after treatment with PM since studies have reported choline as an indispensable extracted from dried apricots.23 nutrient where human with deprivation of choline were seen to develop either fatty liver disease and induce Prunus mume has reported to have documented evidence hepatic cell death and also found to have significant of improving liver function either in alcoholic, non- skeletal muscle damage.20 Clinical evidences have alcoholic liver disease, hepatic fibro-proliferative disease, reported that patients who are on total parenteral chronic hepatitis and overall damaged hepatic health.24 Its nutritional support having low choline was found to activity involves hepato-protective activity, anti-oxidant/ develop fatty liver as well as liver damage. Researches anti-inflammatory activity, increasing HDL cholesterol as have reported that there exists two main source of human well as improving glycemic levels, thereby elevating the dietary requirement of choline: status of estrogen and overall function of liver and preventing metabolic and variations in genetics.21 Through endogenous inflammatory-based disorders.25 biosynthesis of hepatic phosphatidylcholine, dietary requirement of choline can be spared. Gene for Several mouse models have reported choline phosphatidylcholine synthesis is expressed and induced administration in non-alcoholic fatty liver disease have by estrogen therefore in pre-menopausal and post- reported significant improvements in liver function menopausal women treated with estrogen; dietary status.26,27 Low choline is vital in non-alcoholic fatty liver requirements of choline are diminished. On the other disease pathophysiology since it can disturb hand, some females have genetic polymorphism that mitochondrial functioning as well as fatty acid oxidation. makes them unresponsive to estrogen and they require Deficiency of choline causes alterations in mitochondrial higher levels of choline daily, similar to men.22 composition and decreases breakdown products of choline thereby reducing ATP production in The results of our study also reported similar findings to mitochondria of rate given choline deficient diets.28 both Prunus mume and choline as reported in the Similarly, in our study, a significant improvement was literature, where significant decrease in levels of total observed when choline was given to the patients having bilirubin (p=0.04), ALT (p<0.01) and AST were reported altered levels of liver function tests.

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Limitations of the study 7. Yan XT, Lee SH, Li W. Evaluation of the antioxidant and anti-osteoporosis activities of Although this study evaluated substantial data regarding chemical constituents of the fruits of Prunusmume. safety and efficacy of Prunus mume and choline, Food Chem 2014;156:408-15. however the study was not immune from selection and 8. Khan A, Pan JH, Cho S, Lee S, Kim YJ, Park YH. observer bias. Due to the fact that they study was Investigation of the hepatoprotective effect of conducted on a limited sample size, therefore further PrunusmumeSieb. etZucc extract in a mouse model larger studies are needed to evaluate the safety and of alcoholic liver injury through high-resolution efficacy of Prunus mume and choline on altered ALT and metabolomics. J Med Food. 2017;20(8):734-43. AST levels. 9. Zhang Q, Chen W, Sun L, Zhao F, Huang B, Yang W, et al. The genome of Prunusmume. Nature CONCLUSION Communications. 2012;3:1318-26. 10. Jin HL, Lee BR, Lim KJ, Debnath T, Shin HM, Lim This study reported significant improvement in alanine BO. Anti-inflammatory effects of Prunusmume transaminase, aspartate aminotransferase and gamma- mixture in colitis induced by dextran sodium sulfate. glutamyl transferase levels after treatment with Prunus Korean J Med Crop Sci. 2011;19(1):16-23. mume and choline (revolic). The most common side 11. Sherriff JL, O'Sullivan TA, Properzi C, Oddo JL, effect that was observed was gastrointestinal disturbance Adams LA. Choline, its potential role in of minor nature. Therefore, it is safe and effective to use nonalcoholic fatty liver disease, and the case for them for abnormal liver functions tests. human and bacterial genes. Adv Nutr. 2016;7(1):5- 13. ACKNOWLEDGEMENTS 12. Mokhtari Z, Gibson DL, Hekmatdoost A. Nonalcoholic fatty liver disease, the gut We are thankful to Muhammad Shakil for providing microbiome, and diet. Adv Nutr. 2017;8(2):240-52. logistic support of this research project. 13. Perumpail BJ, Cholankeril R, Yoo ER, Kim D, Ahmed A. An overview of dietary interventions and Funding: No funding sources strategies to optimize the management of non- Conflict of interest: None declared alcoholic fatty liver disease. Diseases. 2017;5(4):23- Ethical approval: The study was approved by the 34. Institutional Ethics Committee 14. B Hollenbeck C. An introduction to the nutrition and metabolism of choline. Central Nervous System REFERENCES Agents Med Chem. 2012;12(2):100-13. 15. Fagone P, Jackowski S. Phosphatidylcholine and the 1. Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, CDP-choline cycle. Biochim Biophys Acta. Henry L, Wymer M. Global epidemiology of 2013;1831(3):523-32. nonalcoholic fatty liver disease—meta‐analytic 16. Beretta A, Accinni R, Dellanoce C, Tonini A, assessment of prevalence, incidence, and outcomes. Cardot JM, Bussière A. Efficacy of a Standardized Hepatology. 2016;64(1):73-84. Extract of Prunus mume in Liver Protection and 2. Younossi ZM, Stepanova M, Afendy M, Fang Y, Redox Homeostasis: A Randomized, Double‐Blind, Younossi Y, Mir H, et al. Changes in the prevalence Placebo‐Controlled Study. Phytotherapy Res. of the most common causes of chronic liver diseases 2016;30(6):949-55. in the United States from 1988 to 2008. Clin 17. Xia D, Shi J, Gong J, Wu X, Yang Q, Zhang Y. Gastroenterol Hepatol. 2011;9(6):524-30. Antioxidant activity of Chinese mei (Prunusmume) 3. Chalasani N, Younossi Z, Lavine JE, Charlton M, and its active phytochemicals. J Med Plants Res. Cusi K, Rinella M, et al. The diagnosis and 2010;4(12):1156-60. management of nonalcoholic fatty liver disease: 18. Hokari A, Ishikawa T, Tajiri H, Matsuda T, Ishii O, practice guidance from American Association for Matsumoto N, et al. Efficacy of MK615 for the the Study of Liver Diseases. Hepatology. treatment of patients with liver disorders. World J 2018;67(1):328-57. Gastroenterol. 2012;18(31):4118-26. 4. Cichoż-Lach H, Michalak A. Oxidative stress as a 19. Yan XT, Lee SH, Li W, Sun YN, Yang SY, Jang crucial factor in liver diseases. World J HD, et al. Evaluation of the antioxidant and anti- Gastroenterol. 2014;20(25):8082-91. osteoporosis activities of chemical constituents of 5. Hassan HA, El-Gharib NE, Azhari AF. Role of the fruits of Prunusmume. Food Chem. natural antioxidants in the therapeutic management 2014;156:408-15. of hepatocellular carcinoma. Hepatoma Res. 20. Caballero F, Fernandez A, Matias N. Specific 2016;2:216-3. contribution of methionine and choline in nutritional 6. Mitani T, Horinishi A, Kishida K. Phenolics profile nonalcoholicsteatohepatitis: impact on of mume, Japanese apricot (Prunus mume Sieb. mitochondrial S-adenosyl-L-methionine and etZucc.) fruit. Biosci Biotechn olBiochem glutathione. J Biol Chem. 2010;285:18528-36. 2013;77:1623.

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